Enterohemorrhagic E. coli (EHEC) O157:H7 produces the phage-encoded toxin Stx, which can be absorbed from the intestine and cause life-threatening hemolytic uremic syndrome (HUS), featuring anemia, thromobcytopenia and renal failure. The absorption of Stx across the intestinal barrier is poorly understood. Given that the epithelial cytoskeleton is integral to the regulation of transcytotic and paracellular transport, Stx translocation may be facilitated by the ability of EHEC to dramatically alter host filamentous (F-) actin. EHEC, along with enteropathogenic E. coli (EPEC) and the natural mouse pathogen Citrobacter rodentium, trigger the formation of attaching and effacing (AE) lesions on epithelial cells. These lesions are characterized by intimate attachment and rearrangement of F-actin to form "pedestals" beneath bound bacteria. The ability to generate AE lesions requires the type III translocation of bacterial effector proteins, and the effectors Tir and EspFU have been implicated in the formation of actin pedestals. In addition, EspFU can promote the disruption of epithelial barriers in vitro. We identified the activities of Tir and EspFU required for pedestal formation and have isolated numerous mutants that are incapable of promoting localized actin assembly. Recently, to examine the pathogenesis of Stx-mediated disease, we generated a murine model for EHEC infection utilizing C. rodentium lysogenized with a phage encoding Stx2, an allele of Stx particularly associated with HUS. Mice infected with C. rodentium (?Stx2) suffer Stx-dependent renal damage, weight loss and eventual death. We have also constructed derivatives of C. rodentium (?Stx2) that express EHEC virulence factors, including Tir and/or EspFU, and a pilot experiment was suggestive that the ability to generate cytoskeletal changes may enhance virulence in this model. To test the hypothesis that EHEC mediated actin rearrangement contributes to the disruption of intestinal epithelial barrier function and enhancement of Stx-mediated disease, we will pursue the following aims: 1.Assess the ability of EHEC-mediated actin rearrangement to enhance Stx2 translocation across polarized epithelial cells in vitro. Polarized monolayers will be infected with (a non-Stx- producing) EHEC expressing wild type Tir and EspFU, or with derivatives that are incapable of triggering actin rearrangement. Purified Stx2d will be added to the apical surface and its translocation across infected monolayers of T84 intestinal epithelial cells will be measured. 2. Assess the ability of EspFU to enhance Stx2-mediated disease in a mouse model for EHEC. Mice will be orally infected with C. rodentium (?Stx2) expressing wild type TirEHEC and EspFU, or with derivatives of these effectors that specifically lack activities required for triggering actin rearrangement. Bacterial colonization, intestinal permeability, and systemic disease will be assessed. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in North America, Europe and Japan (79). The life-threatening systemic manifestations of infection are due to the absorption of bacterial toxin from the intestine (51, 52). EHEC binds to cells of the gut wall and triggers the host cell to generate striking pedestal-like structures beneath bound bacteria (33), a process that may promote the absorption of toxin into the mammalian bloodstream. These studies investigate whether the ability of EHEC to induce pedestals promotes systemic disease.